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Published on: July 9, 2014
Targeted data-independent acquisition for mass spectrometric detection of RAS mutations in formalin-fixed,
Yeoun Jin Kim1, Andrew G Chambers1, Fabiola Cecchi1
1Nantomics, LLC, 9600 Medical Center Dr., Rockville, MD 20850, United States.
Abstract:
Genomic testing for KRAS and NRAS mutations in clinical biopsies of various cancers is routinely performed to predict futility of anti-epidermal growth factor receptor (anti-EGFR) therapies. We hypothesized that RAS mutations could be detected and quantified at the protein level for diagnostic purposes using data-independent acquisition (DIA)-based mass spectrometry in formalin-fixed, paraffin-embedded (FFPE) tumor samples. We developed a targeted DIA assay that surveys the specific mass range of all possible peptides harboring activating mutations in KRAS exon 2. When the assay was applied to tumor samples with known KRAS or NRAS mutations (G12A, G12D, G12V, and G13D), RAS-mutant and wild-type peptides were successfully detected in 11 of 13 biopsy samples. Mutation statuses obtained by DIA were concordant with those obtained by DNA sequencing, and yields of mutant peptide (mutant peptide/[mutant + wild-type peptides]) exhibited linear correlation with yields of RAS-mutant mRNA. When applied to biopsy samples with failed DNA testing results, the DIA assay identified an additional RAS-mutated sample. SIGNIFICANCE: Proteomic detection of RAS mutations by DIA in tumor biopsies can provide solid evidence of mutant RAS protein regardless of the mutation types and sites in exon 2. This robust method could rescue samples that fail genomic testing due to insufficient tumor tissue or lack of sequenceable DNA. It can be used to explore the relationship between protein expression level of mutant RAS and therapeutic outcome.
Insights
Proteomic testing using data-independent acquisition mass spectrometry can detect RAS mutations in tumor biopsies. This method complements genomic testing, identifying mutations missed by DNA sequencing.
Area of Science:
- Oncology
- Proteomics
- Molecular Diagnostics
Background:
- Genomic testing for KRAS and NRAS mutations is crucial for predicting response to anti-EGFR therapies in cancer.
- Current methods rely on DNA sequencing, which can fail due to insufficient tumor tissue or DNA quality.
Purpose of the Study:
- To develop and validate a targeted data-independent acquisition (DIA) mass spectrometry assay for detecting and quantifying RAS mutations at the protein level in FFPE tumor samples.
- To assess the concordance of DIA-based proteomic mutation detection with traditional DNA sequencing methods.
Main Methods:
- Development of a targeted DIA mass spectrometry assay to survey peptides harboring activating mutations in KRAS exon 2.
- Application of the assay to formalin-fixed, paraffin-embedded (FFPE) tumor samples with known KRAS/NRAS mutations.
- Comparison of mutation status determined by DIA with results from DNA sequencing.
Main Results:
- The DIA assay successfully detected RAS-mutant and wild-type peptides in 11 of 13 tested biopsy samples.
- Mutation status determined by DIA was concordant with DNA sequencing results.
- The assay identified an additional RAS-mutated sample from biopsies that failed DNA testing, demonstrating its utility in challenging samples.
- Yields of mutant peptides showed a linear correlation with RAS-mutant mRNA levels.
Conclusions:
- Proteomic detection of RAS mutations via DIA mass spectrometry in tumor biopsies offers a robust alternative and complement to genomic testing.
- This method can successfully identify RAS mutations regardless of type or location within exon 2.
- The DIA assay can rescue samples unsuitable for genomic testing and has potential for exploring the relationship between mutant RAS protein levels and therapeutic outcomes.
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